The Dpy-30 domain resembles, in sequence as well as structure, the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase (PKA). The history of RIIa and the emerging evidence on proteins containing the Dpy-30 domain can attest the importance of the discoveries related to the Dpy-30 domain pertain to this application.
RIIa targets PKA to A-Kinase-Anchoring-Proteins (AKAPs) in various micro-compartments in cells. The precise localization of PKA is central for this critical yet promiscuous enzyme that regulates various cellular reactions. Mutations interfering with the localization and function of PKA have severe impact on health and longevity. Various PKA-based reagents including those perturbing the binding between RIIa and AKAPs have been successfully developed into research tools and have potentials to be converted into therapeutic products. RIIa binds to a ˜14-18-a.a. amphipathic helix (AH) in AKAPs. Some AHs has higher affinity than the others. Those AHs with a higher affinity block those with lower affinity. The first-generation AHs were further modified to generate additional AHs of varying affinity and specificity to different isoforms of PKA. In practice, the peptides, modified to improve solubility and membrane permeability, were used in at least two ways. For research, the high-affinity peptides perturb the binding in a RIIa overlay assay and the perturbation is necessary to confirm novel AKAPs. They were shown to perturb cellular reactions. In addition, they have been used in the discovery of non-peptidic chemical blockers.
In the same vein, the binding peptides to the Dpy-30 domain will become powerful tools. Dpy-30, as RIIa, is present in a handful of important molecules, most of which have not been vigorously studied. Yet it has been demonstrated that Dpy-30 protein, from which the domain derives its name, plays critical roles in development and in health. It is present in a wide range of organisms from single cell organisms to human. It is a key subunit in a major group of chromosome modification complexes—Set-1 like histone methyltransferase complexes. In nematodes, the body of the Dpy-30 mutant appears dumpy. In mammals, the chromosome modification complexes are the key culprits in acute mixed lineage lymphoma (MLL). The enzyme, Dpy-30 specifically, is crucial for the embryonic stem cells to differentiate into neurons. As shown from the history of PKA and RIIa, the discovery of high-affinity ligand for Dpy-30 will stimulate research in many directions, both basic and clinical.
In 2009 and 2010, it was demonstrated that human Dpy-30 protein binds to Ash2L of Set1-like histone methyltransferase complex and BIG1 of Golgi apparatus. And the binding fragments were identified. Thus like RI or RII of PKA, Dpy-30 as well as the Dpy-30 domain, likely have many binding proteins in the cells. However, the regions are longer than the binding cavity of the Dpy-30 domain. Furthermore, it has not been demonstrated that the interaction is through the Dpy-30 domain and the common features of the binding peptides remain unknown. Yang lab recently discovered the binding peptides respectively for RIIa domains and Dpy-30 domains in a highly conserved protein, RSP3 in the flagella of Chlamydomonas—a green alga and in humans. In particular, the binding site was narrowed down to 18-25 a.a. This small peptide in algal and human RSP3 share common features with each other and with the AHs for RIIa domains. Both peptides are amphipathic helices and thus are named AHR after the AH that bind RIIa in cells and AHD after the AH that bind Dpy-30 in cells. In addition, based on the study of flagellar protein, we discovered the shortest region in human Ash2L that bind Dpy-30 domain in the histone methyltransferase complex. However, there are distinctions between AHD and AHR.
This study defines peptides that will bind the Dpy-30 domain specifically. Importantly, the AHR binds RIIa domains as well as Dpy-30 domains in vitro. The cross reactivity of AHR suggests that the applications using AHs that have been discovered so far potentially will cause serious problems. In contrast, AHDS from flagellar RSP3 and from Ash2L are monospecific and highly selective to the Dpy-30 domain, indicating a high specificity. Furthermore, the interaction appears to be of high affinity because of the constitutive interactions of the Dpy-30 domains with RSP3 in flagella. Furthermore, the interaction can resist up to 0.6 M KI a chaotropic salt. These results strongly indicate that the Dpy-30-domain-binding peptides from RSP3 and from Ash2L could be modified into high-affinity and high-specificity derivatives that can be used to perturb the interaction of the Dpy-30 domain in methyltransferase complexes and in various circumstances in or outside the cells. The blocking peptides could be used for a wide range of applications, as shown in the blocking peptides for AKAP.